The present invention relates to methods for biopreparation of cellulosic fibers, particularly textiles and most particularly cotton fabrics, at high temperatures using thermostable pectate lyases.
An important aspect of the preparation of textiles from cellulosic fibers is the removal of non-cellulosic components found in the native fiber, as well as the removal of impurities, such as compounds added to the fiber as sizing and lubricants used in the processing machinery. The removal of non-cellulosic impurities, termed xe2x80x9cscouringxe2x80x9d, optimally results in a fabric with a high and even wettability that, consequently, can be evenly bleached and/or dyed.
Conventional scouring processes typically utilize highly alkaline chemical treatment, which results not only in removal of impurities but also in weakening of the underlying cellulose component of the fiber or fabric. Furthermore, chemical scouring creates environmental problems in effluent disposal, due to the chemicals employed and the materials extracted from the fibers. Consequently, there is a need in the art for scouring methods that are specifically targeted to removal of impurities and that are environmentally friendly.
Enzymatic scouring of textiles has been performed using multicomponent fungal enzyme systems comprising pectinases and cellulases that are active at a pH of about 4-5 (Bach et al., Textilveredlung 27:2, 1992; Bach et al., Textilpraxis International, March 1993, p. 220-225; Rxc3x6ssner, Melliand Textilberichte 2: 144, 1993; Rxc3x6ssner, Textilveredlung 30:82, 1995; Hardin et al., 1997 Proceedings Beltwide Cotton Conferences, pp. 745-747; Li et al., Textile Chemist and Colorist 29:71, 1997; Li et al., 1997 International Conference and Exhibition (AATCC), pp. 444-454). In these studies, only a small proportion of the total enzyme activity in the preparations is useful for scouring. These methods thus require the use of large amounts of the enzyme preparation, making them economically unfeasible. Bacterial pectinases, sometimes combined with hemicellulases such as arabinanase, have also been used; these enzymes are typically active at higher pHs (International Patent Application WO9802531; Sakai et al., Textile Engineering (in Japanese), 45:301, 1992; Japanese patent 6220772; Sakai, Dyeing Industry (in Japanese) 43:162, 1995). All reported bacterial pectinases, however, require divalent cations for activity and are not generally active at temperatures over 60xc2x0 C. These properties limit their application to bioscouring of textiles, since (i) the textiles must be pre-boiled to attenuate the waxy cuticle overlaying the pectin layer and (ii) calcium ions tend to form insoluble salts which precipitate on the surface of the fibers.
Thus, there is a need in the art for bioscouring methods that can be performed in a single step, at temperatures near or above the melting temperature of the waxy cuticle of cotton (70xc2x0 C.) and in the absence of added divalent cations, using enzymes that effectively remove pectin and thereby facilitate the removal of pectin and other non-cellulosic impurities.
The present invention provides methods for treating cellulosic fibers to remove non-cellulosic compounds. The methods are carried out by contacting the fibers with an enzyme having pectin-degrading activity, preferably pectate lyase activity, at high temperatures, under conditions that result in pectin removal. Preferably, at least about 30% by weight of the pectin in the fibers is removed; more preferably, at least about 50%, and most preferably, at least about 70%, is removed. The contacting is preferably performed at a temperature above about 70xc2x0 C.; most preferably, above about 80xc2x0 C. In preferred embodiments, the contacting is performed (i) at a pH of at least about 7; more preferably, at least about 8; and most preferably, at least about 9; and (ii) in the absence of added divalent cations.
Pectin-degrading enzymes useful for practicing the invention include without limitation those that (i) exhibit maximal pectate lyase enzymatic activity at a temperature above about 70xc2x0 C., preferably above about 80xc2x0 C.; (ii) exhibit maximal activity at a pH above about 8, preferably above about 9; and (iii) exhibit enzymatic activity that is independent of the presence of divalent cations. It will be understood that any pectate lyase may be used that is sufficiently active above about 70xc2x0 C. to remove at least about 30% by weight of the pectin in the fiber.
In one series of embodiments, the methods use a thermostable pectate lyase comprising a polypeptide having at least 70% homology to the amino acid sequence of SEQ ID NO:1. In preferred embodiments, the thermostable pectate lyase comprises the amino acid sequence of SEQ ID NO:1. See, e.g., Example 2 below. The plasmid comprising DNA encoding SEQ ID NO:1 has been transformed into a strain of E. coli and a bacterial clone containing the plasmid was deposited according to the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on Sep. 8, 1998, under deposit number DSM 12404.
In another series of embodiments, the methods use a pectate lyase comprising a polypeptide having at least 70% homology to the amino acid sequence of SEQ ID NO:2 of co-pending U.S. patent application Ser. No. 09/073,684, filed May 6, 1998. See, e.g., Example 2 below.
Pectate lyases for use in the present invention are preferably derived from Bacillus species, more preferably from B. licheniformis, B. agaradhaerens, B. alcalophilus, B. pseudoalcalophilus, B. clarkii, B. halodurans, B. lentus, B. clausii, B. gibsonii, or related Bacillus species. Variant pectate lyases derived from any pectate lyase polypeptide may also be used in practicing the invention, so long as they exhibit thermostable pectate lyase enzymatic activity, which is preferably alkaline and/or divalent cation-independent.
The methods of the invention can be used for treating crude fibers, yarn, or woven or knit textiles. The fibers may be cotton, linen, flax, ramie, rayon, or blends of these fibers with each other or with other natural or synthetic fibers. The non-cellulosic compounds that are removed using the methods of the invention may be compounds derived from the fiber or compounds derived from manufacturing processes, such as, e.g., spinning, coning, or slashing lubricants.
In some embodiments, the invention further comprises contacting the fibers with one or more other enzymes, including, without limitation, proteases, pectin-degrading enzymes, and lipases.
In another aspect, the invention provides a method for textile preparation which comprises subjecting the textile to simultaneous or sequential (i) scouring and (ii) bleaching, wherein the scouring comprises contacting the textile with an enzyme having thermostable pectate lyase activity, under conditions that result in removal of at least about 30% by weight of the pectin in the textile. In some embodiments, the scouring and bleaching steps are performed simultaneously. The textile may also be subjected to desizing, dyeing, and/or biopolishing using other enzymes.
The present invention provides advantages over conventional scouring processes, including: (i) shorter processing times; (ii) more efficient emulsification and removal of waxes; and (iii) full compatibility with existing state-of-the-art textile processing technologies, including, e.g., continuous pad steam systems.